Coronary angioplasty results in successful nonsurgical revascularization in more than 90% of patients. More than 300,000 coronary angioplasty procedures were performed in the United States in 1990. However, the major limitation of coronary angioplasty is a 30-40% restenosis rate which occurs in the first six months following the procedure.
Vascular smooth muscle cells (SMC) have been identified as playing an important role in the development of atherosclerosis and restenosis following coronary angioplasty. Their presence has been confirmed in both types of lesions, and is due primarily to a change from a contractile to a synthetic phenotype of SMCs. This remarkable characteristic is associated with SMC proliferation, migration from media to intima, and the synthesis of extracellular matrix, all of which results in neointimal formation (narrowing of the artery). In contrast to atherosclerosis, where this process is extended over several decades, vascular restenosis represents an acute response to balloon injury culminating in a significant renarrowing by neointimal formation of an initially patent vessel in the course of a few months. Hence, it has become apparent that the inhibition of SMC growth is necessary to control the restenosis process.
Intensive experimental and clinical investigation for the prevention of restenosis has been conducted over the past decade. Several interventions, such as anti-platelet, anti-coagulation, anti-inflammatory and vasodilator therapies have shown favorable reduction in the severity of neointimal proliferation following experimental balloon injury. Powell et al., Science 1989, 245, 186-188; Castellot, J. et al., J. Cell Physiol. 1985, 124, 21-38; Fox et al., Science 1988, 241, 453-456.
Recently, attempts have also been made to apply new mechanical devices to limit restenosis (e.g., stent, atherectomy, laser, rotablator, etc.). However, preliminary data showed a limited role of these interventions because while mechanical interventions improve the primary result of coronary angioplasty, the mechanical techniques extend vessel wall injury related to the procedure and are therefore unable to reduce SMC proliferation and the restenosis rate. Furthermore mechanical interventions can be applied only to a small group of patients with optimal coronary anatomy.
Application of antimitogenic therapy has also been suggested for prevention of restenosis. For example, concentrated heparin has been tested as an antiproliferative agent to control the problem of restenosis after angioplasty. Wolinsky and Thung, JACC 1990, 15(2), 475-481. .gamma.-interferon has been identified as another potentially useful therapeutic for treatment of restenosis. WO 90/03189 issued Apr. 5, 1990. However the dose of antiproliferative agents given by systemic administration is likely not high enough to achieve the desired effect. Therefore, agents which have been tested are not powerful enough to show a beneficial effect in more complex clinical situations.
Recent advances in cellular and molecular biology have provided insight into the molecular mechanisms of SMC proliferation which is due to the transduction of signals from the extracellular environment (e.g., growth factors) to the cell nucleus. Several genes become transiently activated during phenotypic modulation of SMCs (Gabbiani et al., J Clin Invest 1984, 73, 148-152; Walker et al., Proc Natl Acad Sci USA 1986, 83, 7311-7315; Miano et al., Am J Pathol 1990, 137, 761-765; Majesky et al., Circulation Research 1992, 71, 759-768). These findings have stimulated interest in defining the role of abnormal gene expression in SMC growth and in selecting potential therapeutic targets for molecular-based approaches for acquired cardiovascular disorders such as vascular restenosis. Recently, Speir and Epstein, Circulation 1992, 86, 538-547, showed the growth inhibition of rat smooth muscle cells using high concentrations of antisense oligonucleotide complementary to proliferating cell nuclear antigen mRNA.
Nuclear proto-oncogenes are highly conserved phosphoproteins tightly linked to cellular proliferation. The transient increase in nuclear proto-oncogene (s) mRNA following mitogenic stimulation has been shown as the cell enters the G1 phase and appears to be necessary for the G1-to-S phase transition. Studies in cultured SMCs have demonstrated that c-fos, c-myc, and c-myb proto-oncogenes are activated shortly after various mitogenic stimuli (Kindy et al., J Biol Chem 1986, 261, 12865-12868; Brown et al., J Biol Chem 1992, 267, 4625-4630). Proto-oncogene expression has also been induced in the vessel wall following balloon denudation in a pattern similar to in vitro studies (Miano et al., Am J Pathol 1990, 137, 761-765). The above observations and the redundancy of signal transduction pathways have raised the possibility that nuclear proto-oncogene activation is a final common pathway onto which many diverse mitogenic signals converge, making it a potential therapeutic target. Collins et al. J. Clin. Invest. 1992, 89, 1523-1527 found that antisense oligonucleotides complementary to c-myc inhibited the colony formation of colonic carcinoma cells. Other groups have focused on the proto-oncogene, c-myb and have found that inhibition of c-myb inhibits the proliferation of smooth muscle cells. Recently, Simon and Rosenberg, Circulation Research 1992, 70, 835-843 showed the growth-inhibitory effect of c-myb antisense oligonucleotides in rat smooth muscle cells.
Antisense technology is emerging as an effective means of lowering the levels of a specific gene product. It is based on the findings that these "antisense" sequences hybridize a gene or associated target polynucleotide, to form a stable duplex or triplex, based upon Watson-Crick or Hoogsteen binding, respectively. The specifically bound antisense compound then either renders the respective targets more susceptible to enzymatic degradation, blocks translation or processing, or otherwise blocks or inhibits the funciton of a target polynucleotide. Where the target polynucleotide is RNA, the antisense molecule hybridizes to specific RNA transcripts disrupting normal RNA processing, stability, and translation, thereby preventing the expression of a targeted gene. Administration of antisense oligonucleotides or transfer of expression constructs capable of producing intracellular antisense sequences complementary to the mRNA of interest have been shown to block the translation of specific genes in vitro and in vivo. For example, Holt et al., Mol. Cell. Biol. 1988, 8, 963-973, focusing on c-myc, found the formation of an intra-cellular duplex with target mRNA and a selective decrease of c-myc protein in human promyelocytic leukemia HL-60 cells.
Methods of modulating the proliferation of smooth muscle cells associated with restenosis in any vascular bed is greatly desired. Such method should be applicable to patients having a broad range of vascular disorders including coronary and peripheral stenoses (i.e., blockages). Further, the method should have high efficacy. Such methods are provided by the present invention.